脂多糖诱导肿瘤坏死因子释放因子基因过表达慢病毒载体的构建及鉴定

探讨构建人脂多糖诱导肿瘤坏死因子释放因子(LITAF)基因的过表达慢病毒载体的技术方法,并检测其体外表达目的基因的水平。设计LITAF基因引物,应用聚合酶链反应(PCR)的方法扩增LITAF基因片段;应用EcoRI、Bam HI酶切LV8载体,通过连接酶将LITAF基因片段连接至线性化的LV8载体上,应用酶切及测序方法鉴定LITAF-LV8重组质粒,将其包装慢病毒后感染293T细胞(人胚肾细胞),观察绿色荧光蛋白(GFP)的表达,验证后感染肝癌细胞株HepG2。RT-PCR和Westernblot鉴定感染后HepG2中LITAF的表达。LITAF基因在慢病毒感染的HepG2细胞中的表达显著高于对照组细胞。说明成功构建了过表达LITAF的HepG2细胞株,为后期研究提供了实验基础。     

稳定表达Cytoglobin的肝细胞株LO-2的建立及生长增殖的变化

目的:建立过表达细胞珠蛋白Cytoglobin(CYGB)的人肝细胞LO-2稳定株,初步探究CYGB对LO-2细胞生长增殖的影响.方法:采用PCR法扩增CYGB编码基因,并通过GATEWAY克隆技术构建慢病毒重组表达载体Plenti-N-GFP-CYGB;酶切、测序鉴定后,与包装质粒三质粒共转人肾上皮细胞(HEK293T),收集、浓缩含病毒上清,获得病毒颗粒;感染LO-2细胞并采用流式细胞分选技术筛选稳定细胞株,Western blot检测表达情况;使用CCK-8试剂盒检测过表达CYGB对LO-2细胞增殖的影响,通过流式细胞技术检测过表达CYGB对LO-2细胞凋亡的影响.结果:慢病毒重组表达载体Plenti-N-GFP-CYGB双酶切鉴定以及基因序列比对鉴定均正确.三质粒共转HEK293T细胞后,荧光显微镜下观察大部分细胞出现绿色荧光;收集病毒颗粒并感染LO-2细胞后,经流式细胞分选技术筛选获得稳定表达CYGB的细胞,Western blot鉴定显示,LO-2稳定株组的CYGB表达条带明显,阴性对照组未出现条带.CCK-8法绘制的细胞生长曲线显示,稳定表达细胞组的细胞生长速率低于阴性对照组,有统计学差异(P0.01).流式细胞技术检测各组LO-2细胞凋亡情况结果显示,稳定表达细胞组的细胞凋亡百分比高于阴性对照组(P0.05).结论:成功构建慢病毒重组表达载体Plenti-N-GFP-CYGB;建立稳定表达CYGB的人肝细胞株LO-2-GFP-CYGB以及阴性对照组细胞株LO-2-GFP;稳定表达CYGB的肝细胞生长速率较阴性对照组肝细胞降低,稳定表达CYGB的肝细胞凋亡百分比高于阴性对照组肝细胞;为CYGB与其蛋白的相互关系研究提供了细胞模型.     

携人源TPA基因的慢病毒表达质粒的构建与鉴定

利用PCR扩增人源TPA基因,再用酶切-连接的方法将目的片段亚克隆入慢病毒表达质粒pL-PGK-eGFP中,最后用测序、酶切和在293细胞中瞬时表达的方法进行了鉴定.结果显示：含人源TPA基因的pL-PGK-TPA-eGFP慢病毒表达载体构建成功,为进一步利用转基因技术更加安全高效地生产TPA,治疗血栓类疾病打下基础.     

含PGK启动子慢病毒表达质粒的构建与鉴定

为了构建一个含有人源PGK1(human phosphoglycerate kinase 1)启动子的慢病毒表达载体 pL-PGK-GFP.采用PCR从人组织中扩增PGK1基因的启动子部分,再用酶切-连接的方法将扩增的启动子区片段亚克隆入慢病毒表达质粒pL-EGFP中,再用测序、酶切和瞬时表达的方法进行鉴定.结果是下游的eGFP基因在PGK1启动子驱动下,在293FT细胞中表达绿色荧光蛋白报告基因,这表明成功构建了慢病毒表达质粒pL-PGK-GFP.扩增的537bp PGK1启动子片段具有一定的启动效率,能在HIV来源的慢病毒载体中驱动下游目的基因的表达.     

基于Cre-LoxP系统的新型慢病毒表达载体pLOX-CMV-E/P的构建与鉴定

目的:基于Cre-Lox P系统构建携带EGFP及Puromycin抗性基因,并带有巨细胞病毒(CMV)及翻译延伸因子1α(EF1α)双启动子的新型慢病毒表达载体,为基因治疗及基因功能研究提供有效的慢病毒载体系统.方法:以已经插入Lox P序列的p LOX-TERT-ires TK慢病毒载体为模板,用Spe I与Kpn I进行双酶切,去除TERT-ires TK片段,然后与人工设计合成的多克隆位点片段连接,构建p LOX-MCS表达载体.同时,以p B513载体为模板扩增EF1α-EGFP-Puro表达框(带有Bam HI及Kpn I酶切位点),然后与经过Bam HI及Kpn I双酶切的p LOX-MCS载体连接,进而构建p LOX-CMV-EF1α-EGFP-Puro(简称p LOX-CMV-E/P)载体.将p LOX-CMV-E/P载体与慢病毒包装载体p CMVR8.74及p MD2.G共转染293T细胞,包装病毒进行报告基因的功能分析.结果:菌落聚合酶链式反应(PCR)、酶切鉴定及测序结果均与预期结果一致,绿色荧光蛋白及抗药性基因均有较好的活性与功能.结论:成功构建了p LOX-MCS及p LOX-CMV-E/P慢病毒表达载体,为基因治疗及基因功能研究提供有效的慢病毒表达系统.     

小鼠Sirt3基因过表达慢病毒载体的构建及其表达鉴定

构建小鼠Sirt3基因过表达慢病毒载体,检测人神经母细胞瘤细胞(SK-N-SH细胞)感染Sirt3基因过表达慢病毒后Sirt3 mRNA和蛋白的表达,为在细胞和动物水平研究Sirt3的作用提供新的途径和工具。利用Genebank检索的小鼠Sirt3基因序列,人工合成法合成小鼠Sirt3基因的c DNA片段,将其克隆至慢病毒载体p CDH-CMV-MCS-EF1-cop GFP中,构建慢病毒表达质粒。进行酶切及测序验证后,将慢病毒表达质粒连同辅助包装原件载体质粒共同转染入293T细胞,收集上清,测定病毒滴度。用Sirt3基因过表达慢病毒感染SK-N-SH细胞,即为Sirt3过表达组(Sirt3);用空载体慢病毒感染的SK-N-SH细胞为空载体组(GFP);未感染病毒的SK-N-SH细胞为对照组(CON)。收集细胞后,用Real-time PCR法检测各组细胞Sirt3 mRNA的水平;用Western blotting法检测各组细胞Sirt3蛋白的表达。Sirt3基因过表达慢病毒载体构建成功,病毒滴度为1.0×10~9TU/m L。Sirt3组SK-N-SH细胞的Sirt3 mRNA和Sirt3蛋白水平均显著高于GFP组和CON组(P0.01)。成功构建的Sirt3基因过表达慢病毒载体具备高效感染力,能在SK-N-SH细胞高效过表达Sirt3,本研究结果为今后进一步在神经细胞和模型动物中研究Sirt3的作用提供了新的途径和工具。     

干涉UBXN1慢病毒颗粒制备及稳定细胞系的建立

构建并鉴定UBXN1-shRNA慢病毒表达载体,以便应用RNAi技术以及慢病毒感染系统建立稳定干涉细胞系并进一步研究UBXN1的功能。将携带不同特异性干涉序列的DNA片段插入PLKO.1载体中构建慢病毒表达载体,并制备慢病毒颗粒。将慢病毒颗粒感染U2OS细胞,建立稳定干涉细胞系,应用real-time PCR和western blot技术分别检测U2OS细胞中UBXN1 mRNA和蛋白质水平的表达差异。重组克隆经酶切证实shRNA正确插入慢病毒载体,DNA测序证实插入的序列正确,western blot检测证实设计的五条shRNA干扰序列有效的敲低U2OS细胞中内源性UBXN1的表达。成功制备UBXN1的慢病毒干涉颗粒,并建立UBXN1稳定下调的U2OS细胞系。     

DLL3蛋白在人小细胞肺癌细胞中的功能及其机制

为探讨DLL3(human notch ligand delta-like 3)过表达对人小细胞肺癌细胞的影响及可能的作用机制,通过PCR方法扩增人DLL3基因全长序列,并克隆至慢病毒表达载体Lenti-EFS-FLAG-puro而构建人DLL3基因过表达慢病毒表达质粒,酶切及测序鉴定质粒正确。通过慢病毒包装及感染,构建DLL3稳定过表达的人小细胞肺癌细胞株,并通过Western blot验证DLL3蛋白的表达。采用CCK-8法检测DLL3过表达对人小细胞肺癌细胞的增殖的影响。采用平板克隆实验检测DLL3过表达对人小细胞肺癌细胞的克隆形成的影响。Western blot检测DLL3过表达对细胞周期相关蛋白Cyclin D1,Cyclin D3的表达水平的影响。结果表明:人DLL3过表达慢病毒表达质粒构建成功; CCK-8实验显示DLL3过表达促进人小细胞肺癌细胞的增殖。平板克隆实验显示DLL3过表达提高人小细胞肺癌细胞的克隆形成能力。Western blot结果表明DLL3过表达增加细胞周期蛋白Cyclin D1,Cyclin D3的表达水平。可见DLL3过表达对人小细胞肺癌细胞的增殖具有促进作用。     

MiR-218慢病毒表达载体的构建及鉴定

摘要:构建人mir-218-2, pre-miRNA慢病毒表达载体,为研究miR-218在人体的功能及作用机制打下基础。以人hsa-mir-218-2前体序列,设计部分互补的正反向引物,进行引物退火,形成引物二聚体,PCR扩增引物二聚体,酶切后插入到线性化pGCSIL-GFP慢病毒表达载体中,对重组质粒进行双酶切鉴定,并进行慢病毒的包装与滴度检测。用构建好的慢病毒表达载体感染人胃癌细胞MKN-28,qPCR检测细胞内miR-218表达。结果显示,重组质粒经双酶切分析及转化菌液测序,插入序列正确,慢病毒表达载体感染人胃癌细胞后qPCR检测显示能显著增高miR-218的表达。说明本实验成功构建了hsa-mir-218-2慢病毒表达载体,感染人胃癌细胞后能有效提高miR-218的表达。为进一步研究miR-218在人体的功能及作用机制建立了实验基础。     

Characterization of Ordovician carbonate reservoirs, southeastern Saskatchewan, Canada

The discovery of the prolific Ordovician Red River reservoirs in 1995 in southeastern Saskatchewan was the catalyst for extensive exploration activity which resulted in the discovery of more than 15 new Red River pools. The best yields of Red River production to date have been from dolomite reservoirs. Understanding the processes of dolomitization is, therefore, crucial for the prediction of the connectivity, spatial distribution and heterogeneity of dolomite reservoirs.The Red River reservoirs in the Midale area consist of 3～4 thin dolomitized zones, with a total thickness of about 20 m, which occur at the top of the Yeoman Formation. Two types of replacement dolomite were recognized in the Red River reservoir: dolomitized burrow infills and dolomitized host matrix. The spatial distribution of dolomite suggests that burrowing organisms played an important role in facilitating the fluid flow in the backfilled sediments. This resulted in penecontemporaneous dolomitization of burrow infills by normal seawater. The dolomite in the host matrix is interpreted as having occurred at shallow burial by evaporitic seawater during precipitation of Lake Almar anhydrite that immediately overlies the Yeoman Formation. However, the low δ18O values of dolomited burrow infills (-5.9‰～ -7.8‰, PDB) and matrix dolomites (-6.6‰～ -8.1‰, avg. -7.4‰ PDB) compared to the estimated values for the late Ordovician marine dolomite could be attributed to modification and alteration of dolomite at higher temperatures during deeper burial, which could also be responsible for its 87Sr/86Sr ratios (0.7084～0.7088) that are higher than suggested for the late Ordovician seawaters (0.7078～0.7080). The trace amounts of saddle dolomite cement in the Red River carbonates are probably related to "cannibalization" of earlier replacement dolomite during the chemical compaction.     

A computer generator for randomly layered structures

AcomputergeneratorforrandomlylayeredstructuresYUJia shun1,2,HEZhen hua2(1.TheInstituteofGeologicalandNuclearSciences,NewZealand;2.StateKeyLaboratoryofOilandGasReservoirGeologyandExploitation,ChengduUniversityofTechnology,China)Abstract:Analgorithmisintrod…     

An Index mechanism for multi-scale view in spatial databases

There are numerous geometric objects stored in the spatial databases. An importance function in a spatial database is that users can browse the geometric objects as a map efficiently. Thus the spatial database should display the geometric objects users concern about swiftly onto the display window. This process includes two operations:retrieve data from database and then draw them onto screen. Accordingly, to improve the efficiency, we should try to reduce time of both retrieving object and displaying them. The former can be achieved with the aid of spatial index such as R-tree, the latter require to simplify the objects. Simplification means that objects are shown with sufficient but not with unnecessary detail which depend on the scale of browse. So the major problem is how to retrieve data at different detail level efficiently. This paper introduces the implementation of a multi-scale index in the spatial database SISP (Spatial Information Shared Platform) which is generalized from R-tree. The difference between the generalization and the R-tree lies on two facets: One is that every node and geometric object in the generalization is assigned with a importance value which denote the importance of them, and every vertex in the objects are assigned with a importance value,too. The importance value can be use to decide which data should be retrieve from disk in a query. The other difference is that geometric objects in the generalization are divided into one or more sub-blocks, and vertexes are total ordered by their importance value. With the help of the generalized R-tree, one can easily retrieve data at different detail levels.Some experiments are performed on real-life data to evaluate the performance of solutions that separately use normal spatial index and multi-scale spatial index. The results show that the solution using multi-scale index in SISP is satisfying.     

海南岛地体及其毗邻陆缘晚中生代—新生代古地磁研究和构造演化

本文叙述了对海南岛及其毗邻大陆边缘白垩纪到第四纪地层岩石进行古地磁研究的全部工作过程。通过分析岩石中剩余磁矢量的磁偏角及磁倾角的变化,提出海南岛白垩纪以来经历的构造演化模式如下:早期伴随顺时针旋转而向南迁移,后期伴随逆时针转动并向北运移。联系该地区及邻区的地质、地球物理资料,对海南岛上述的构造地体运动提出以下认识:北部湾内早期有一拉张作用,主要是该作用使湾内地壳显著伸长减薄,形成北部湾盆地。从而导致了海南岛的早期构造运动,而海南岛后期的构造运动则主要是受南海海底扩张的影响。海南地体运动规律的阐明对于了解北部湾油气盆地的形成演化有重要的理论和实际意义。     

Exciton Seebeck in Molecular Systems

Various applications relevant to the exciton dynamics,such as the organic solar cell,the large-area organic light-emitting diodes and the thermoelectricity,are operating under temperature gradient.The potential abnormal behavior of the exicton dynamics driven by the temperature difference may affect the efficiency and performance of the corresponding devices.In the above situations,the exciton dynamics under temperature difference is mixed with     

Achieving Unusual States of Matter Under High Pressure

The elongation method,originally proposed by Imamura was further developed for many years in our group.As a method towards O(N)with high efficiency and high accuracy for any dimensional systems.This treatment designed for one-dimensional(ID)polymers is now available for three-dimensional(3D)systems,but geometry optimization is now possible only for 1D-systems.As an approach toward post-Hartree-Fock,it was also extended to     

《Chinese Science Bulletin》SUBJECT INDEX TO VOLUME 59(2014)

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An on-line scaling method for improving scalability of a database cluster

The explosive growth of the Internet and database applications has driven database to be more scalable and available, and able to support on-line scaling without interrupting service. To support more client's queries without downtime and degrading the response time, more nodes have to be scaled up while the database is running. This paper presents the overview of scalable and available database that satisfies the above characteristics. And we propose a novel on-line scaling method. Our method improves the existing on-line scaling method for fast response time and higher throughputs. Our proposed method reduces unnecessary network use, i.e. , we decrease the number of data copy by reusing the backup data. Also, our on-line scaling operation can be processed parallel by selecting adequate nodes as new node. Our performance study shows that our method results in significant reduction in data copy time.     

An improved R-tree based on childnode''s probability

R-Tree is a good structure for spatial searching. But in this indexing structure,either the sequence of nodes in the same level or sequence of traveling these nodes when queries are made is random. Since the possibility that the object appears in different MBR which have the same parents node is different, if we make the subnode who has the most possibility be traveled first, the time cost will be decreased in most of the cases. In some case, the possibility of a point belong to a rectangle will shows direct proportion with the size of the rectangle. But this conclusion is based on an assumption that the objects are symmetrically distributing in the area and this assumption is not always coming into existence. Now we found a more direct parameter to scale the possibility and made a little change on the structure of R-tree, to increase the possibility of founding the satisfying answer in the front sub trees. We names this structure probability based arranged R-tree (PBAR-tree).     

Integrating GIS Web services based on mediating architecture

The geographic information service is enabled by the advancements in general Web service technology and the focused efforts of the OGC in defining XML-based Web GIS service. Based on these models, this paper addresses the issue of services chaining,the process of combining or pipelining results from several interoperable GIS Web Services to create a customized solution. This paper presents a mediated chaining architecture in which a specific service takes responsibility for performing the process that describes a service chain. We designed the Spatial Information Process Language (SIPL) for dynamic modeling and describing the service chain, also a prototype of the Spatial Information Process Execution Engine (SIPEE) is implemented for executing processes written in SIPL. Discussion of measures to improve the functionality and performance of such system will be included.